Although weight gain and obesity are nearly universal features of Cushings syndrome, we hypothesized that screening results would have poor specificity in an obese population. The study is based in a weight loss clinic and enrolled individuals with at least two additional signs or symptoms of Cushings syndrome. 369 subjects (73% female) completed two or three tests: a 24h urine cortisol (UFC), and/or late-night salivary cortisol, and/or 1 mg dexamethasone suppression test (DST). If any result was abnormal (based on laboratory reference range or cortisol after DST >/= 1.8 ug/dl 50 nmol/l), tests were repeated and/or a dexamethasone-CRH (dex-CRH) test was performed. Subjects with abnormal DST results and a low dexamathasone level were asked to repeat the test with 2mg of dexamethasone. We found that in addition to obesity, subjects had a mean of 5-6 features of Cushing's syndrome. None was found to have Cushing's syndrome. Test specificities to exclude Cushing's syndrome for subjects who completed 3 tests were: UFC 96% 95 CI: 93-98%; DST 90% 95 CI: 87-93%; salivary cortisol 84% by RIA 95 CI: 79-89% and 92% by LC-MS/MS 95 CI: 88-95%. The combined specificity (both tests normal) for all combinations of two tests was 84 to 90%, with overlapping confidence intervals. These data do not support widespread screening of overweight and obese subjects for Cushing's syndrome; test results for such patients may be falsely abnormal. Within the same population we have evaluated the influence of cortisol on parameters of the metabolic syndrome.UFC and dexamethasone responses were not associated with BMI or weight. However, salivary cortisol showed a trend to increase as BMI increased (P < 0.0001), and correlated with waist circumference (WC) in men (rs = 0.28, P = 0.02) and systolic BP in women (rs = 0.24, P = 0.0008). Post-dexamethasone cortisol levels were weak to moderately correlated with fasting insulin (rs = -0.31, P = 0.01) and HOMA-IR (rs = -0.31, P = 0.01) in men and systolic (rs = 0.18, P = 0.02) and diastolic BP (rs = 0.20, P = 0.009) in women. PSS results were higher in obese subjects than controls, but were not associated with cortisol or metabolic parameters. As expected, WC correlated with fasting insulin, HOMA-IR, and systolic BP (adjusted for BMI and gender; P < 0.01). Literature showed inconsistent relationships between cortisol and metabolic parameters.